17 allylamino 17 demethoxygeldanamycin 17 aag

U-87 MG and M059K cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Taastrup, Denmark) supplemented with 10% fetal bovine serum (FBS, Biochrom AG, Berlin, Germany) at 37°C under a 5% CO₂ atmosphere. Cells were treated with 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG, Sigma-Aldrich, Schnelldorf, Germany), 1,3-Dichloro-6-[(E)-((4-methoxyphenyl)imino)methyl] diben- zo(b,d) furan-2,7-diol, referred to as D11 (DTP, NIH/NCI, Rockville, MD, USA), MG132 (Sigma-Aldrich), CX-4945 (Selleck Chemicals, Houston, TX, USA), 4-[(E)-(fluoren-9-ylidenehydrazinylidene)-methyl] benzoic acid (referred to as E9, DTP, NIH/NCI, USA), Glutamine (Gln, Sigma-Aldrich) and Bafilomycin A1 (Sigma-Aldrich) as indicated in the figure legends. Cells were cultured in Gln-free medium for 12 h prior incubation with 2 mM Gln for 12 h. Down-regulation of protein kinase CK2 and HSP70 was carried out by small interfering RNA (siRNA, Dharmacon, CO, USA) essentially as described in [18 (link)]. The WST-1 assay (Roche, Hvidovre, Denmark) was performed to measure cell viability determined in a microtiter plate reader according to the manufacturer’s recommendations (Perkin-Elmer, Waltham, MA, USA).

To generate polyclonal antibodies to TRABID, two TRABID fragments, corresponding to the three Npl4-like zinc finger domains (3NZF; residues 1–200) and the ankyrin-repeat domain (Ank; residues 260-340) were cloned to pET32a to generate 6XHis-tagged recombinant proteins. The recombinant proteins were purified using Ni Sepharose (GE Healthcare) under denaturing conditions and used as antigens. Antiserum production and affinity purification were performed by LTK BioLaboratories (Taipei, Taiwan). Other antibodies used in this study are described in the Supplementary Data 2. Oleic acid, Doxycycline, Bafilomycin A1, Cycloheximide, 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) and 17-Desmethoxy-17-N,N-dimethylaminoethylamino-geldanamycin (17-DMAG) were purchased from Sigma-Aldrich, whereas tunicamycin and thapsigargin were obtained from Cayman Chemical. MG132 was purchased from Calbiochem, and rapalog was from Clontech. Puromycin was obtained from Gibco. VPS34 inhibitor (VPS34i; SAR405) was purchased from MedChemExpress.

Mouse monoclonal antibodies (mAbs) against RABV N or P were prepared in our laboratory46 (link). The rabbit polyclonal antibody (pAb) against GAPDH was purchased from Hangzhou Good Here Biotechnology Co. Ltd (Hangzhou, China). Rabbit anti-ß-actin and anti-Myc pAbs were obtained from Hangzhou Huaan Biotechnology Co. Ltd (Hangzhou, China). Rabbit anti-Hsp90AA1, anti-Cdc37 (phospho S13) and anti-Cdc2 mAbs were purchased from Abcam (Cambridge, MA, USA). Rabbit anti-LC3B, anti-Cdc37 and anti-IKKa mAbs were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-Flag (clone M2) mAb was purchased from Sigma-Aldrich (St. Louis, MO, USA). Alexa Fluor 546-conjugated donkey anti-mouse IgG and Alexa Fluor 647-conjugated goat anti-rabbit IgG were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The proteasome inhibitor MG-132 and the de novo protein synthesis inhibitor cycloheximide (CHX) were purchased from Beyotime Biotechnology (Shanghai, China). The Hsp90 inhibitor 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), the Hsp90 allosteric inhibitor celastrol and the autophagy inhibitor wortmannin were purchased from Sigma.